Sprayable tissue adhesive with biodegradation tuned for prevention of postoperative abdominal adhesions

Abstract Adhesions are dense, fibrous bridges that adjoin tissue surfaces due to uncontrolled inflammation following postoperative mesothelial injury. A widely used adhesion barrier material in Seprafilm often fails to prevent transverse scar tissue deposition because of its poor mechanical properties, rapid degradation profile, and difficulty in precise application. Solution blow spinning (SBS), a polymer fiber deposition technique, allows for the placement of in situ tissue‐conforming and tissue‐adherent scaffolds with exceptional mechanical properties. While biodegradable polymers such as poly(lactic‐co‐glycolic acid) (PLGA) have desirable strength, they exhibit bulk biodegradation rates and inflammatory profiles that limit their use as adhesion barriers and result in poor tissue adhesion. Here, viscoelastic poly(lactide‐co‐caprolactone) (PLCL) is used for its pertinent biodegradation mechanism. Because it degrades via surface erosion, spray deposited PLCL fibers can dissolve new connections formed by inflamed tissue, allowing them to function as an effective, durable, and easy‐to‐apply adhesion barrier. Degradation kinetics are tuned to match adhesion formation through the design of PLCL blends comprised of highly adhesive “low”‐molecular weight (LMW) constituents in a mechanically robust “high”‐molecular weight (HMW) matrix. In vitro studies demonstrate that blending LMW PLCL (30% w/v) with HMW PLCL (70% w/v) yields an anti‐fibrotic yet tissue‐adhesive polymer sealant with a 14‐day erosion rate countering adhesion formation. PLCL blends additionally exhibit improved wet tissue adhesion strength (~10 kPa) over a 14‐day period versus previously explored biodegradable polymer compositions, such as PLGA. In a mouse cecal ligation model, select PLCL blends significantly reduce abdominal adhesions severity versus no treatment and Seprafilm‐treated controls.


| INTRODUCTION
Abdominal adhesions are deposits of dense, connective scar tissue that form between organ surfaces as a result of uncontrolled fibrogenesis following surgery, trauma, inflammation, infection, or tissue ischemia. 1,2 Such uncleaved fibrous bridges are frequently reported in the human peritoneum following surgical interventions resulting in broad serous tissue injury (e.g., abrasion, suturing), and are particularly common following abdominal surgeries such as laparotomy and appendicectomy. 3,4 Pathologic adhesion formation takes place due to an imbalance between the early fibrin deposition and degradation that occurs as part of healing after trauma, as well as the proximity of an injured surface to other structures. [5][6][7] In normal abdominal tissue healing, the entire injured surface heals uniformly, and affected cells secrete numerous pro-inflammatory cytokines, growth factors, and coagulants such as fibrin. Fibrous matrix deposition begins within 3 hours of tissue injury and increases until postinjury Day 4 or 5, where it is then enzymatically degraded through fibrinolysis over the course of 1 week. In postsurgical adhesion formation, fibrin deposition outpaces fibrinolysis during the healing process and permanent connective adhesions are created between organs, with up to 93% of patients developing adhesions following operation in the abdomen or pelvis. [8][9][10] Such unsuppressed proliferation of fibrous tissue frequently causes small bowel obstruction, female infertility, or chronic abdominal or pelvic pain and is implicated in up to 60%, 40%, and 80% of cases, respectively. [11][12][13][14] Removal of postsurgical adhesions through adhesiolysis can be attempted laparoscopically as to reduce frequency and severity in the abdominal cavity, but ultimately these procedures only have a~70% success rate while also increasing the risk of new adhesion formation. 15 Treatment of small bowel obstruction accounts for up to 1% of all general surgical admissions, 3% of all laparotomies, over $2 billion in hospitalization and surgical expenditures annually, as well as an approximate 900,000 days of inpatient care. 10,[16][17][18] Because these surgical interventions to treat adhesions prove to be largely ineffective and costly, prophylactic barrier materials are needed that can prevent adhesions between organs before they form. Hydrogel-based adhesion barriers are the most widely adopted tool in surgical settings, but are difficult to apply, poorly adhesive to the target organ, and degrade too quickly to effectively prevent adhesions. 13 Currently available clinical products to prevent adhesion formation include Seprafilm (Genzyme)-a predried hydrogel film made of carboxymethylcellulose-hyaluronic acid that swells once in contact with aqueous abdominal fluid-and Interceed (Johnson & Johnson), a woven cellulose mat. Both products act as solid barriers and physically prevent adhesions by separating injured mesothelial surfaces through interfacial lubrication imparted by their hydrophilic surface properties.
Because they are prefabricated, such clinical products are brittle and difficult to apply, with limited flexibility when conforming to geometrically complex tissue surfaces. They also degrade rapidly in moist environments in the critical 5-day maturation period for adhesions, exhibit impeded wound healing, and inability to seal sites of injury, the combination of which limits their use in clinical practice. [19][20][21][22][23][24] Furthermore, Seprafilm undergoes a 90% loss in tensile strength within 30 minutes due to swelling of its carboxymethylcellulose-derived network, which renders it largely ineffective in the abdominal cavity where organs are in perpetual motion and tissue surfaces are routinely extending. 19,20 Recent biomaterials research efforts have recently focused on use of physically crosslinked hydrogels comprised of nanoparticles dispersed in a cellulose matrix. 25,26 However, they exhibit reduced flexibility and adherence to wet tissue, and also require an intricate syringe-based deposition technique. Other investigated hydrogel systems include ones forming chemical crosslinks to tissue in situ via reactive end group chemistries, as the resultant material mimics biological tissue stiffness and thereby promotes biocompatible interfacing upon implantation. [27][28][29] However, such materials frequently swell, causing undue pressure on surrounding tissue, and utilize crosslinking approaches that employ either toxic initiators or adhesive curing processes such as ultraviolet light and high temperature. 30,31 An implanted material for use as an adhesion barrier must not only be retained at the sight of application, but also maintain mechanical integrity during critical stages of fibrosis and wound healing.
To develop an adhesion barrier that is sprayable, tissue adhesive to only the target organ, degradable at the same rate as the abdominal tissue wound healing process, and does not impede wound healing, we investigated solution blow spinning (SBS) of dry, conformal polymer fibers with controllable surface erosion. Through blending of fast degrading low-molecular weight (LMW) and slow degrading highmolecular weight (HMW) surface eroding polymers at defined ratios, we can design sprayable fiber mats with linear biodegradation profiles tuned to a clinically relevant rate. Previous research investigations from our group have reported the biocompatibility and efficacy of SBS-deposited polymer materials for in vivo surgical applications including antimicrobial burn wound dressings, 32 sealants for intestinal anastomosis, [33][34][35] and hemostats for traumatic bleeding. 36 While stretchy, durable materials are desirable for high tissue adhesion, viscoelasticity, and tunable biodegradation are necessary to provide a matrix that facilitates complete wound healing in a moist environment. For example, cohesively strong poly(lactic-co-glycolic acid) (PLGA) not only displays a lack of wet tissue adherence unless blended with an additional adhesive component, but also induces abdominal adhesions in a clinical mouse model over a 10-day time course. 37 Such shortcomings are a result of a near 0% loss in polymer mass and remaining polymer providing a template for fibrous tissue growth. In contrast, separate biodegradable viscoelastic polymer blends were investigated as the primary dressing in a porcine partialthickness wound model, exhibiting high wet tissue adherence (>1 N/cm 2 ) and complete wound healing in a pressure-sensitive tissue adhesive (PSTA) application. 37,38 In this report, we studied the effect of various molecular weight blends of poly(lactide-co-caprolactone) (PLCL) on biodegradation profile, cohesive strength, and tissue adhesion, followed by implementation into a preclinical mouse model of abdominal adhesions. Multiple LMW and HMW combinations of PLCL were studied to modulate surface erosion rate and determine its subsequent effect on adhesion prevention. Since degraded fragments of PLCL continually erode from the surface, PLCL has the potential to act as a favorable adhesion barrier material if its degradation profile is tuned to coincide with adhesion formation (Figure 1). A constantly eroding surface will mitigate cell adhesion and fibrin deposition, which are necessary steps for the formation of adhesions. 39,40 The barrier itself-tuned to retain the necessary mechanical properties at the application site-is critical to occlude atypical deposition of fibrous, vascular scar tissue until the target wound itself has healed. We aimed to strike a balance between the presence of critical wound healing components through kinetic control of degradation, as well as necessary cohesive and adhesive strength through facile tuning of HMW and LMW ratios. PLGA was referenced as a bulk degrading control that undergoes minimal ero-   (a) Formation of adhesions is a consequence of reduced fibrinolytic activity following ischemic mesothelial tissue injury, (b) leading to deposition of connective wound healing tissue. (c) Our poly(lactide-co-caprolactone) (PLCL) molecular weight blends yield a viscoelastic, wet tissue adhesive rapidly deposited via solution blowspinning (SBS) for application and retention in the abdominal cavity, while also presenting a surface erosion degradation mechanism apt to (d) prevention of adhesion formation and (e) wound healing Adhesions form within 5-7 days and then mature over 2 weeks.
Any potential barrier material needs to prevent contact between surfaces during the initial stages of fibrin deposition and persist until the injured mesothelium is healed. Blending HMW 40k or 80k PLCL with LMW 5k PLCL at a 70:30 ratio results in a linear degradation profile for up to 14 days (~50% mass loss) ( Figure 2b) while also displaying distinct bimodal molecular weight distributions in GPC not presented in other blends and neat compositions (Figures 2c,d, S1). As these particular blends begin to degrade and decrease in molecular weight, there is a shift to a unimodal distribution with a high PDI (~3) due to the presence of 5k PLCL as synthesized, along with degraded portions of 40k or 80k PLCL. Since all other blend compositions yield only 5%-20% mass loss and plateau in later stages, 40k/5k and 80k/5k PLCL blends have the potential to equivalently release short-chain fragments from the polymer surface over a 14-day treatment period for adhesions. The fast, linear erosion rate will decrease the accumulation of fibro-and angio-genic molecules, such as fibrinogen and vascular endothelial growth factor (VEGF), thereby reducing scar tissue formation on healing mesothelium.
Blending either LMW 5k or 20k PLCL in an HMW 40k or 80k PLCL matrix greatly promotes tensile elasticity as both are near (20k) or below (5k) entanglement molecular weight, while also presenting viscous behavior that permits flow upon the application of an external force. Both 40k/5k and 80k/5k PLCL blends in particular display improved adhesive strength to tissue versus their neat 40k or 80k PLCL compositions, as the 5k component allows the sealant to spread across a given surface under application of pressure. Adhesion to a surface under these conditions is facilitated through physical mechanisms of polymer chain entanglement with complex tissue topography and short-range interactions (e.g., Van der Waals) with surface molecules as facilitated through the viscoelastic nature of our adhesive. [41][42][43] As expected, blending 5k or 20k PLCL produces materials with decreased stiffness (Figure 3a) and values of yield stress ( Figure S2 Blending of different molecular weights allows for tunable degradation with rapid linear degradation in the first several days for 80k/5k and 40k/5k blends. HMW = "high" molecular weight. LMW = "low" molecular weight. ( 0 ) = HMW peak of blend. ( 00 ) = LMW peak of blend. Data are plotted as mean ± SE *p < 0.05; **p < 0.01; ***p < 0.001

| In vivo efficacy in a mouse model of abdominal adhesions and wound healing
Below entanglement molecular weight polymers (~1 kDa) formed in vivo can exhibit toxic effects due to an ability to disrupt cell membrane integrity. 44 We therefore assessed toxicity prior to in vivo implantation of 5k PLCL in either neat or blend compositions. L929 mouse fibroblasts were treated with supernatant of degraded polymer. Neat 5k PLCL significantly reduced cell viability (~50%) of L929 mouse fibroblasts at Â1 concentration, while neat 40k and blended 40k/5k PLCL compositions had no effect on cell viability at all dilutions ( Figure 4a). This indicates that 40k/5k PLCL blends have low toxicity and could be safely used as an implanted adhesion barrier material.
An accurate in vivo animal model for adhesion formation should produce consistent and reproducible mesothelial injury and ischemia.
Forceful abrasion of serosal tissue lining the abdominal cavity and cecal ligation have been previously used to induce adhesions. 45 Although more directly related to operative conditions, abrasion models are largely subjective as the amount of force applied by the operator can vary. Therefore, a cecal ligation mouse model was Since fibrin deposition and remodeling is a process regulated by pro-inflammatory signaling molecules, 46,47 cecal tissue was extracted from the mice at Day 7 for analysis of gene expression and histology.
Histologic evidence of inflammation, which coincides with adhesion formation, or healing can be used to corroborate assessments of F I G U R E 3 (a) Tensile stiffness, (b) yield strain, and (c) day 0 pull-apart adhesion strength for (i) band-aid-to-skintissue and (ii) cardiac-patch-to-intestinetissue of neat and blend poly(lactide-cocaprolactone) (PLCL) during in vitro degradation. MW, molecular weight. Both pull-apart adhesion tests were done with 1 min of applied pressure, as to show the positive effect on tissue adherence with blending 20k or 5k PLCL. Data is plotted as mean ± SE. Asterisks indicate statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001 adhesion score severity. Hematoxylin and eosin (H&E) stained cecum displayed infiltration of neutrophils and eosinophils throughout the entire intestinal wall in all cecal ligation groups (Figures 5a,b, S4).
Quantitative measurements of gross inflammation further assessed via cellularity analysis did not demonstrate significant differences between saline, Seprafilm, and polymer groups, although all cecal ligation groups displayed increased cellularity compared with the "no surgery" group, as expected.   ., 30%). This ratio was selected as to remain in a similar material regime of published work using PLCL molecular weight blends for pressure sensitive tissue adhesive (PSTA) applications where multiple ratios were studied. 37,38 An airbrush (Master Airbrush, G222-SET, 0.2 mm nozzle diameter) was used to deposit the solutions as dry, conformal polymer fibers.
The airbrush was connected to a compressed CO 2 tank equipped with a pressure regulator set to 20 psig.

| Tensile strength testing
Tensile strength testing was performed to determine the mechanical properties of the polymer samples over time. For the 0-day (i.e., nondegraded) experiment, samples were produced by spraying 2 mL of polymer solution onto a glass coverslip. For 1, 3, 7, and 14-day timepoints, polymer samples were degraded according to the procedure described in the degradation testing section, removed from the coverslips, and trimmed to a rectangular shape approximately 10 mm by 5 mm in size. Exact sample dimensions were measured immediately prior to testing. Tensile testing was performed on a TA Instruments DMA Q800 equipped with a film tensile clamp. Samples were stretched under a controlled force ramp from 0 to 5 N at a rate of 0.01 N/min and measurements made at room temperature. Elastic modulus was calculated from the linear region of the resulting stress versus strain curve, with a 0.2% offset used to calculate sample yield stress and strain. Each sample type was replicated 5 times (n = 5).

| Pull-apart adhesion testing
Pull-apart testing was performed on a TA Instruments DMA Q800.

| Cell viability
Cytotoxicity of polymer compositions was tested against L929 mouse fibroblasts by an elution method as described by ISO-10993-5. 49 40k/5k PLCL blend and neat 40k PLCL and 5k PLCL compositions were sprayed onto sterile 22 mm by 22 mm glass coverslips. The polymer mats were then removed from the coverslips and eluted at mass concentration of 10 mg/ml in culture media of Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (Gemini Bio-Products Inc.), L-glutamine and 1% penicillin and streptomycin at standard conditions (37 C, 5% CO 2 ) for 24 h. The elutions were diluted to Â1, Â10, and Â100 dilutions, and cell viability was tested against the different dilutions.

| Statistical analysis
Statistical analysis was performed on Origin (OriginLab). Typically, one-way ANOVA was used to compare group variation, followed by post hoc pairwise Tukey comparison to determine significant differences between the groups. Typically, averages were plotted with error bars representing standard error (SE). Asterisks are used to indicate statistically significant differences: *p < 0.05, **p < 0.01, ***p < 0.001.
If no asterisks are shown, there are no significant differences among the groups. Real-time PCR results were analyzed using t-tests comparing the ΔΔCt values.

CONFLICT OF INTEREST
The authors have no conflicts of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.